Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corre- sponding quinone, L-dopaquinone. In spite of its biomedi- cal relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the pro- tein, improving cell culture conditions, and setting a suit- able purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharma- ceutical or cosmetic agents addressing tyrosinase activity.

Human Tyrosinase Produced in Insect Cells: A Landmark for the Screening of New Drugs Addressing its Activity

FOGAL, STEFANO;CAROTTI, MARCELLO;GIARETTA, LAURA;LANCIAI, FEDERICO;NOGARA, LEONARDO;BUBACCO, LUIGI;BERGANTINO, ELISABETTA
2014

Abstract

Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corre- sponding quinone, L-dopaquinone. In spite of its biomedi- cal relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the pro- tein, improving cell culture conditions, and setting a suit- able purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharma- ceutical or cosmetic agents addressing tyrosinase activity.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3041977
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